Methylphenidate enhances neuronal differentiation and reduces proliferation concomitant to activation of Wnt signal transduction pathways

Methylphenidate (Ritalin) is the most commonly prescribed drug in the treatment of attention-deficit hyperactivity disorder. It is suggested that in vivo, methylphenidate treatment supports cortical maturation, however, the molecular and cellular mechanisms are not well understood. This study aimed to explore the potential effect of methylphenidate on cell proliferation and maturation in various cellular models, hypothesizing its interaction with the Wnt-signaling. The termination of cell proliferation concomitant to neuronal maturation following methylphenidate treatment was observed in all of the cell-models tested: murine neural stem-, rat PC12- and the human SH-SY5Y-cells. Inhibition of Wnt-signaling in SH-SY5Y cells with Dkk1 30 min before methylphenidate treatment suppressed neuronal differentiation but enhanced proliferation. The possible involvement of the dopamine-transporter in cell differentiation was discounted following the observation of opposing results after GBR-12909 treatment. Moreover, Wnt-activation via methylphenidate was confirmed in Wnt-luciferase-reporter assay. These findings reveal a new mechanism of action of methylphenidate that might explain long-term effects.


Immunocytochemistry
Fixed and prepared cells were stained for differentiation studies with rabbit monoclonal antibody against glial fibrillary acidic protein (GFAP; 1:200, Sigma aldrich, USA, Cat-No: C4546) and/or with mouse monoclonal antibody against β-tubulin III (Tuj 1; 1:300, Sigma aldrich, USA, Cat-No: T3952) overnight at 4°C. For total cell count, nuclei were visualized using Hoechst 33258 staining (1:100; Invitrogen, Cat-No: H3569) for 15 minutes at room temperature (RT). After overnight incubation, the primary antibodies were visualized with secondary antibodies against rabbit or mouse conjugated to the following fluorochromes: Alexa FluorW -488 and Alexa FluorW -555 (ThermoSchientific, Switzerland). Staining was visualized under the inverted microscope (Olympus IX81, Germany) with DP72 Digital camera and the xcellence software (Olympus, Germany). In Supplementary Fig. S1 examples for staining and overlays are shown.
For each well, first the total number of cells within the well was determined manually counting Hoechst positive stained cells. Secondly, GFAP positive or Tuj 1 positive cells were manually counted, respectively. Subsequently, the relative ratio of positive cells (sum of GFAP or Tuj 1 positive cells, respectively) to the total cell count (sum of Hoechst-stained cells) was calculated. In order to present results in comparison to the untreated cells, percentages were calculated for each treatment dose, setting the control as 100%.

Differentiation of SH-SY5Y and PC12 cells
SH-SY5Y cells were seeded in poly-D-lysine/laminin coated 8-well slides (Corning, Switzerland) at a density of 10 4 cells/well. The cells were grown at 37°C, 5% CO2 for 24 hours in normal growth medium before it was exchanged with a differentiation medium (DM), consisting of a normal growth medium supplemented with 10 μM retinoic acid (Sigma Aldrich, Switzerland) and 50 ng/ml nerve growth factor (Sigma Aldrich, Switzerland). PC12 cells were seeded in Collagen I (ScienCell, USA) coated 4-well slides (Corning, Switzerland) at a density of 10 5 cells/well. The cells were grown at 37°C, 5% CO2 for 24 hours in normal growth medium before it was exchanged with DM, consisting of a 1:5 mixture of normal growth medium and DMEM supplemented with 100 ng/ml nerve growth factor. DM was exchanged every second day.

BrdU Staining
Cells were fixed with 4% ice-cold paraform aldehyde (SantaCruz Biotechnologies, Switzerland) for 20 minutes at RT. This was followed by incubation in 1 M HCl on ice for 10 minutes, followed by 2 M HCl at RT for 20 minutes and another 20 minutes in 2 M HCl at 37°C, in order to enable breaking of the nucleus and denaturing the DNA. After neutralizing with 1.5 M sodium borate buffer for 10 minutes, the cells were washed trice with PBS. To prevent unspecific binding, the cells were blocked in a blocking buffer (PBS, 1% goat serum and 0.03 % triton-X-100) for 90 minutes. Alexa Fluor 488®-linked anti-BrdU antibody (Millipore, Switzerland) was added to the cells, which were then incubated in the dark overnight at 4°C. The cells were washed with PBS three times for five minutes. For the mounting a DAPI (nucleus staining reagent)-containing mounting medium (Abcam, UK) was used. The slide was dried over night before pictures were taken at 20x magnification under the inverted microscope (Olympus IX81, Germany) with DP72 Digital camera. Cells were counted using the xcellence software (Olympus, Germany).

Activity detection of canonical Wnt-signaling with luciferase Wnt reporter assay
To confirm our pharmacological analysis of the Wnt-signaling activation by MPH and R-Spondin1 against Wnt-signaling inhibition via dkk1, we used the Leading Light ® Wnt Reporter Assay (Enzo Life Sciences, Lausen, Switzerland). This assay, is a cell-based luciferase activity test conducted in a 96-well plate, relaying on an engineered 3T3 mouse fibroblast cell line, which expresses the firefly luciferase reported gene under the control of Wnt-responsive promoters (TCF/LEF). Dosedependent up-regulation of luciferase activity can be achieved by adding Wnt protein or Wnt agonist, while down-regulation is achieved by addition of Wnt antagonist. Wnt reporter cell lines were grown in 4500mg/L glucose Dulbecco's modified Eagle medium (DMEM) (Sigma, Switzerland) supplemented with 10% FBS (Thermo Fisher Scientific, Switzerland), 4mM L-Glutamine (Life Technologies) and 1mM Sodium Pyruvate (Life Technologies, Switzerland). 50µl cells (15,000 cells/well) in growth medium were added to the 96-well plate. Plates were incubated overnight in humidified 37 o C and 5% CO2 incubator. Figure S1: Effect of methylphenidate (MPH) on embryonic murine neuronal stem cell (mNSC) differentiation into astrocyte (GFAP + cells) and neurons (Tuj1 + cells). Embryonic mNSC were treated with MPH (1 nM up to 100 µM) or with vehicle (control). The cell number was determined via nucleus staining using Hoechst four days after treatment with MPH, while differentiation into astrocytes was determined by positive staining to the glial fibrillary acidic protein (GFAP) and differentiation into immature neurons was determined by positive staining to the neuron-specific class III beta-tubulin (Tuj1) compared to the total number of cells. Representative immunocytochemistry staining's for each of the treatments and control of embryonic mNSC against GFAP (red), Tuj 1 (green) and for total cell count using Hoechst 33258 staining (blue). Scale bar = 50 µm.